Benefits of HER2-targeted therapy in HER2+ cancers are limited due to significant de novo and acquired resistance, and no therapy options exist for patients with refractory disease. Exploiting oncogene-induced vulnerabilities as a therapeutic strategy in cancers has emerged as a promising alternative to targeting of driver oncogenes. Our preliminary in silico analyses of clinical datasets and in vitro analyses on cell lines have revealed that HER2 overexpression in the mammary epithelial cells induces proteotoxic stress in the endoplasmic reticulum (ER), which is relieved in HER2+ breast cancer cells due to specific overexpression of the ER-associated degradation (ERAD) complex. We have found that HER2+ breast cancer cells are acutely dependent on ERAD to maintain proper protein homeostasis and survival. Accordingly, targeting of ERAD induces proteotoxic ER stress and cell death selectively in EGFR/HER2+ breast cancer cells, including those with de novo and acquired resistance to trastuzumab and lapatinib. In addition, we have found that combinatorial targeting of ERAD and adaptive ER stress response machinery can synergize in the induction of proteotoxic ER stress and cell death in drug- resistant HER2+ cells. Based on these results, we propose that oncogenic HER2-induced proteotoxic ER stress is a clinically significant vulnerability, and that the therapeutic targeting strategies to force irrecoverable proteotoxic ER stress are an effective approach against therapy-refractory HER2+ breast cancers. In this proposal, we propose to 1) identify the mechanisms of addiction of HER2+ cells to ERAD, 2) identify novel potent combinatorial strategies of maximizing oncogenic ER stress, and 3) test in vivo efficacy of inducing oncogenic proteotoxic stress in drug-resistant breast cancers. In Aim 3, we will test several novel inhibitors of ERAD in in vivo tumor growth assays in mice making use of patient- derived xenograft lines.